Exuberant production of pro-inflammatory cytokines has been implicated in the pathogenesis of numerous inflammatory and autoimmune diseases. Secretion of TNFα is a primary event in the initiation of the inflammatory cascade (Brennan F. M., et. al. Lancet, 1989, 2:244-7; Haworth C, et. al. Eur. J. Immunol. 1991, 21:2575-2579) and directly contributes to the initiation and maintenance of these diseases. Other pro-inflammatory cytokines also play a role, including interleukin 1β (IL-1β), IL-6, IL-8, IL-12 nitric oxide (NO), IFN-γ and granulocyte macrophage-colony stimulating factor (GM-CSF), while anti-inflammatory cytokines such as IL-10 may reduce disease. Cells of the immune system, macrophages in particular, secrete many of these cytokines in response to activating stimuli. Blocking pro-inflammatory cytokines and TNFα in particular has been shown to improve the symptoms and progression of a variety of inflammatory diseases. Thus, inhibition of either the production or the activity of TNFα and/or modulation of other cytokines active in the disease is an appealing therapeutic target for the treatment of inflammatory, infectious, autoimmune and other proliferative diseases and conditions.
Many inflammatory diseases have been treated with steroids, methotrexate, immunosuppressive drugs including cyclophosphamide, cyclosporine, azathioprine and leflunomide, nonsteroidal anti-inflammatory agents including aspirin, acetaminophen and cox-2 inhibitors, gold agents and anti-malarials. These drugs have a variety of disadvantages such as adverse reactions and lack of efficacy. New anti-TNFα biologic therapies have emerged that give a faster onset of relief and improved efficacy. However, these protein-based therapies also suffer drawbacks including adverse side effects such as injection site reactions, rash, upper respiratory infections, autoimmune disorders and serious infections. Another shortcoming of these biologic therapies is their required route of administration, intravenous (IV) or subcutaneous (SC), as opposed to more convenient and compliant oral or dermal routes. Accordingly, a need still exists for the development of novel small molecule compositions that inhibit TNFα and/or modulate the expression of one or more other cytokines that can be used to treat inflammatory, infectious, autoimmune and other proliferative diseases and conditions.
The pathogenesis of rheumatoid arthritis (RA), a chronic progressive inflammatory autoimmune disorder, is mediated by cytokines. Several cytokines, including TNFα (Di Giovine F. S., et. al. Ann. Rheum. Dis. 1988, 47:768-772) IL-1 (Rooney M., et. al. Rheumatol Int. 1990, 10:217-219), IL-10 (Arend W. P. et. al. Arthritis. Rheum. 1990, 30:305-315), and GM-CSF (Firestein G. S., et. al. J Exp. Med. 1988, 168:1573-1586.; Xu W. D., et. al. J. Clin. Invest. 1989, 83:876-882), are upregulated in the joints of RA patients with active disease. Elevated levels of TNFα and IL-1β can contribute to joint swelling seen in experimental animal models of arthritis and human RA (Thorbecke, G. J. et. al. Proc. Natl. Acad. Sci., 1992, 89:7375-7379; Chu C. Q., et. al. Arth. Rheum. 1991, 34:1125-1132.; Farahat M. N., et. al. Ann. Rheum. Dis. 1993, 52:870-875; Brennan F. M., et. al. Eur J. Immunol. 1992, 22:1907-1912; Elliott M. J., et. al. Baillieres Clin. Rheumatol. 1995, 9:633-652). Anti-TNFα therapy using both antibodies and fusion proteins against TNFα has been shown to reduce the symptoms of collagen-induced arthritis in mice (Williams, R. O., et. al. Proc. Natl. Acad. Sci., 1992, 89:9784-9788; Wooley P. H., et. al. J Immunol, 1993, 151:6602-6607; Mori L., et. al. J Immunol, 1996, 157:3178-3182) and in human clinical settings (Elliott M. J., et. al. Arth Rheum. 1993, 36:1681-1690; Elliott M. J., et. al. Lancet, 1994, 344:1105-1110; Elliott M. J., et. al. Lancet, 1994; 344:1125-1127; Moreland L. W., et. al. N. Engl J. Med. 1997, 337:141-147; Moreland L. W., et. al. J. Rheumatol. 1996, 23:1849-55; Rankin E. C., et. al. Br. J. Rheumatol. 1995, 34:334-42; Sander O., et. al. Arth. Rheum. 1996, 39 (suppl.):S242 (Abstract)). Recently, anti-IL-1β therapy using a recombinant IL-1 receptor antagonist has been shown efficacious in combination with methotrexate in treating RA patients (Cohen S., et. al. Arth. Rheum. 2002, 46:614-624). In experimental animal models of arthritis, inhibition of NO production (Brahn E., et. al. J. Rheumatol. 1998, 25:1785-1793), blockage of GM-CSF activity (Cook A. D., et. al. Arth. Res. 2001, 3:293-298) or treatment with recombinant IL-10 (an anti-inflammatory cytokine) (Tanaka T., et. al. Inflamm. Res. 1996; 45:283-288) reduced arthritic symptoms. These studies underscore the contribution of individual cytokines to the pathogenesis of RA and imply that therapies that target more than one cytokine or a selective subset of cytokines may be more efficacious.
Therapies that target TNFα have been shown to be effective in other inflammatory diseases in humans and in animal models of human disease. Anti-TNFα therapy has been used to effectively treat Crohn's disease, a chronic inflammatory bowel disease. Infliximab (Remicade, Centocor, Inc.), an intravenously administered monoclonal antibody to TNFα, has been approved for commercial use in refractory Crohn's disease. Mixed results have been seen in patients receiving Etanercept (Enbrel, Amgen Corp.), an injectable TNFα receptor fusion protein. In a small study, patients responded favorably and had decreased inflammatory markers (D'Haens G. et. al. Am J Gastroenterol. 2001, 96:2564-2568) following treatment with this drug. In a different study, Etanercept was found to be ineffective for the treatment of patients with moderate to severe Crohn's disease (Sandborn W. J., et. al. Gastroenterology 2001, 121:1088-1094). The reasons for lack of efficacy could be mechanistic, dose-related or may indicate that the biologic drug did not reach the target cell population. Another TNFα modulator, the small molecule thalidomide, which both down-regulates TNFα production and inhibits angiogenesis, has been shown to relieve experimental iodoacetamide-induced colitis in rats (Kenet G., et. al. Isr. Med. Assoc. J. 2001, 9:644-648). Additionally, early findings in a long-term treatment protocol involving children and young adults treated with thalidomide suggest that treatment resulted in decreased Crohn's disease activity (Facchini S., et. al. J Pediatr. Gastroenterol. Nutr. 2000, 32:178-181).
Spondyloarthropathies (SpA) are a group of related disorders with varying clinical symptoms including spondylitis, synovitis, psoriatic arthritis and subclinical gut inflammation. TNFα appears to play a strong role in the pathogenesis of these syndromes and heightened concentrations of TNFα are found in the joint, skin and gut of patients afflicted with SpA. Etanercept has been approved for treatment of psoriatic arthritis. Infliximab used in two open studies demonstrated significant clinical benefit against SpA (Van den Bosch F., et. al. Ann. Rheum. Dis. 2000, 59:428-433; Baete D., et. al. Arhritis. Rheum. 2001, 44:186-95) and psoriatic arthritis (Ogilvie A. L. et al, Br. J. Dermatol. 2001, 144:587-589). The common mechanism of action of these drugs and their mutual ability to improve clinical outcome suggest that anti-TNFα therapy is useful in the treatment of SpA.
The local release of TNFα contributes to the inflammatory skin disease psoriasis. Concentrations of TNFα and soluble TNFα receptors (p55 and p75) were higher in lesional stratum corneum extracts of psoriatic patients than controls (Ettehadi P., et. al. Clin. Exp. Immunol. 1994, 96:146-151). These results confirm the presence of immunoreactive TNFα and quantifiable concentrations of soluble TNF receptors, which may regulate the effects of TNFα. A number of case reports and small clinical studies indicate that infliximab improved psoriasis symptoms (Chaudhari U., et. al. Lancet 2001, 357:1842-1847; Newland M. R., Int. J. Dermatol. 2002, 41:449-452; Schopf R. E., et. al. J. Am. Acad. Dermatol. 2002, 46:886-91). Etanercept has also been clinically evaluated and shows efficacy against psoriasis symptoms. Biologically active IL-1β has also been found in psoriatic scales (Lundqvist E. N., et. al. Eur. J. Immunol. 1997, 27:2165-2171) suggesting a role for this cytokine in the pathogenesis of psoriasis. Again, the interaction of various cytokines appears to be important in this disease and suggests that efficacious treatments would target a variety of cytokines.
Overproduction of TNFα also contributes to the clinical features of numerous autoimmune diseases such as diabetes. Therapies directed at TNFα have been shown to provide clinical benefit in Type II diabetes. For example, thiazolidinedione derivatives are a class of drugs that activate the peroxisome proliferator-activated receptor-gamma (PPARγ) resulting in diminished macrophage activation and decreased production of TNFα (Ricote M., et. al. Nature 1998, 391:79-82; Jiang C., et. al. Nature 1998, 391:82-86). These drugs have been tested in various human clinical studies in non-insulin-dependent diabetes and these drugs improved control of glucose metabolism and blood lipid profiles (Kumar S., et. al. Diabetologia 1996, 39:701-709; Berkowitz K., et. al. Diabetes 1996, 45:1572-1579; Yamasaki Y. et. al. Tohoku J. Exp. Med. 1997, 183:173-183) in addition to lowering TNFα levels in plasma (Katsuki A., et. al. Diabete. Obes. Metab. 2000, 2:189-2191).
Systemic lupus erythematosus (SLE) is another autoimmune disorder precipitated by increased TNFα levels. Within lupus patients, serum C-reactive protein, IL-1β and TNFα levels were higher than in controls suggesting that an over-exuberant immune response plays a role in the disease (Liou L. B. Clin. Exp. Rheumatol. 2001, 19:515-523). A study of patients with one form of SLE, neuropsychiatric lupus erythematosus (NPLE), showed that the number of peripheral blood mononuclear cells expressing mRNA for TNFα as well as the cerebrospinal fluid level of NO metabolites correlated with NPLE disease severity (Svenungsson E., et al. Ann. Rheum. Dis. 2001, 60:372-9). In a study by Segal, (Segal R., et. al. Lupus 2001, 10:23-31), SLE was induced in mice with human anti-DNA antibodies. These mice were then treated with an anti-TNFα antibody or pentoxifylline, a phosphodiesterase inhibitor that lowers TNFα production. Both treatments reduced the production of TNFα and lowered serum levels of anti-DNA antibodies. The anti-inflammatory cytokine IL-10 has been shown to regulate murine models of lupus. IL-10−/− knockout mice were bred into a lupus susceptible mouse strain. These mice developed more severe lupus and suffered higher mortality than IL-10+/+ mice (Yin Z., et. al. J. Immunol. 2002, 169:2148-2155). An ideal SLE therapy would target multiple cytokines; inhibiting TNFα levels while enhancing or exerting no inhibition on IL-10 levels.
TNFα is also involved in cutaneous forms of lupus. A-308A polymorphism of the human TNFα promoter has a significantly increased prevalence in patients suffering from subacute cutaneous LE. This polymorphism led to substantially higher induction of TNFα after exposure to UVB than wild type, contributing to the photosensitivity seen in this disease (Werth V. P., et. al. J. Invest. Dermatol. 2000, 115:726-730). Biopsies from patients with localized discoid LE showed significantly elevated levels of IL-2 and IFNγ mRNA and elevated levels of TNFα mRNA compared to normal skin (Toro J. R., et. al. Arch. Dermatol. 2000, 136:1497-1501). Overall, these findings suggest that anti-TNFα therapy would benefit patients with these forms of LE.
Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by a T-cell mediated autoimmune response to the myelin sheath. A number of pro-inflammatory cytokines contribute to the ongoing inflammation in human disease (Sharief M. K., et. al. N. Engl. J. Med. 1991, 325:467-472) and murine experimental autoimmune encephalomyelitis (EAE) models (Conlon P., et. al. Neurobiol. Dis. 1999, 6:149-66). Administration of anti-TNFα antibodies in the EAE animal model reversed demyelination and paralysis (Selmaj K., et. al. Ann. Neurol. 1991, 30:694-700; Karin N., et. al. J. Exp. Med. 1994, 180:2227-37). Human trials with monoclonal antibodies and soluble TNFα receptor have been unsuccessful, emphasizing the need to develop new anti-TNFα therapies (van Oosten, B. W. et. al. Neurology 1996, 47:1531-1534).
TNFα has also been found to potently upregulate human immunodeficiency virus 1 (HIV-1) expression in T cell clones (Duh E. J., et. al. Proc. Natl. Acad. Sci. USA 1989, 86:5974-8; Folks T. M., et. al. Proc. Natl. Acad. Sci. USA 1989, 86:2365-2368; Clouse K. A., et. al. J Immunol. 1989, 142:431-8.) and monocytes (Koyanagi Y., et. al. Science 1988, 241:1673-1675). TNFα enhances HIV-1 replication in T cells by increasing the surface density of the HIV docking receptor, CXCR4 (Biswas P., et. al. Cytokine 2001, 13:55-59). In vivo effects of TNFα were demonstrated in a study using homozygous HIV-1 transgenic mice. These mice have significantly increased serum TNFα levels and die within 3-4 weeks. Treatment with an antibody to TNFα prevented death, decreased characteristic skin lesions in these mice, and profoundly reduced HIV-1 expression (De S. K., et. al. J. Virol. 2002, 76:11710-4). Thus, inhibition of TNFα could suppress T cell activation, CXCR4 expression and slow viral spread and replication.
Pro-inflammatory cytokines have been implicated in other viral infections including the cytomegalovirus, influenza virus and the herpes family of viruses. TNFα enhances the basal activity of the major immediate early enhancer/promoter of human cytomegalovirus and may play a role in reactivation of latent HCMV infection in premonocytic cells (Prosch S., et. al. Virology 1995, 208:197-206). Likewise, GM-CSF enhanced de novo influenza A virus protein synthesis, viral particle release and cell death in human monocytes infected by influenza A virus. Treatment of persistently herpes simplex virus (HSV) infected macrophages for 2 weeks with TNFα resulted in an increase of HSV yield and an increase in virus-induced cytotoxic effects (Domke-Opitz I., et. al. Scand. J. Immunol. 1990, 32:69-75). Further studies showed that TNFα and possibly GM-CSF enhanced the reactivation frequency and replication of HSV in the trigeminal ganglia of mice latently infected with HSV (Walev I., et. al. Arch. Virol. 1995, 140:987-992).
A number of cytokines contribute to the demise of patients suffering from sepsis or endotoxic shock. TNFα and IL-1β have a well-established central role in sepsis, septic shock and endotoxic shock. Increased levels of these cytokines are associated with fever, hypotension and shock (Smith J. W. et. al. J. Clin. Oncol. 1992, 10:1141-1152; Chapman P. B., et. al. J. Clin. Oncol. 1987, 5:1942-1951) together with the induction of gene expression for phospholipase A2 (Gronich J., et. al. J. Clin. Invest. 1994, 93:1224-1233) and NO synthase. The induction of NO from smooth muscle cells mediates decreased mean arterial pressure and systemic vascular resistance during septic shock, suggesting a fundamental role for NO. Therapies targeting TNFα in particular with downregulatory effects on IL-1β and NO could be beneficial in the treatment of sepsis, septic shock, and endotoxic shock.
A variety of cell types are involved in the inflammatory process. Overproduction of TNFα by monocytes, macrophages and other immune cells is a key element in the pathogenesis of a multitude of diseases. Macrophages and T-cells in particular play a central role in the initiation and maintenance of the immune response. Once activated by pathological or immunogenic stimuli, macrophages respond by releasing a host of cytokines, including TNFα, IL-1β, IL-8, IL-12, NO, IL-6, GM-CSF, G-CSF, M-CSF and others. T-cells release IL-2, IL-4, interferon-γ, and other inflammatory cytokines. These cytokines activate other immune cells and some can also act as cytotoxic agents alone. Excessive release of macrophage and T-cell derived inflammatory mediators can therefore lead to damage of normal cells and surrounding tissues. The overabundance of these cytokines is a clinical feature of many chronic inflammatory diseases. Treatment and resolution of these conditions may depend on attenuation of the immune cells, particularly the macrophages and T-cells.
Previous reports suggest that polyamines modulate macrophage function by inhibiting the secretion of inflammatory mediators such as TNFα (Zhang M., et. al. J. Exp. Med. 1997, 185, 1759-68). Treatment of human peripheral blood mononuclear cells or a murine macrophage cell line with spermine prior to stimulation with the immunostimulant lipopolysaccharide (LPS) inhibited the release of pro-inflammatory cytokines including TNFα. Spermine treatment had no effect on increases in TNFα mRNA levels, indicating that the suppression occurred post-transcriptionally. In subsequent studies, Zhang et al reported that pretreatment of cells with a polyamine transport inhibitor prior to addition of spermine and LPS restored TNFα levels to those seen in the absence of spermine (Zhang M., et. al. Mol. Med. 1999, 5:595-605). The inhibitory action of spermine may provide a natural mechanism for attenuation of the immune response and protection against excessive inflammatory damage. Furthermore, a U.S. patent (Bergeron, R. J. U.S. Pat. No. 5,843,959) describes bicyclic polyamine compositions that exerted anti-inflammatory effects. These compositions provided modest inhibition of type II collagen-induced arthritis in mice and carrageenan-induced edema in rat paws. An additional U.S. patent (Tracey, K. J. et. al. U.S. Pat. No. 6,482,833) describes spermine antagonists that prevent spermine-induced immunosuppression. Thus the utility of polyamine analog-based therapies in treating inflammatory conditions has been established.